This invention relates to a process for determination of .gamma.-glutamyl transpeptidase activity using .gamma.-L-glutamyl-p-nitroanilide as a substrate.
Determination of .gamma.-glutamyl transpeptiase (hereinafter referred to as ".gamma.-GTP.revreaction.) activity is widely used for clinical diagnosis of hepatoiliary duct disease, screening of drinkers, and the like. Among various methods for determining .gamma.-GTP activity, a method using .gamma.-L-C-glutamyl-p-nitroanilide as a substrate is most popular and employed widely. But .gamma.-L-glutamyl-p-nitroanilide is remarkably poor in solubility at near neutral pH, which is most suitable for the stabilization of substrate and enzymatic reaction. Thus, there is generally employed a so-called acid dissolution method wherein .gamma.-L-glutamyl-p-nitroanilide is sufficiently dissolved at a low pH having relatively higher solubility and mixed with a buffer solution of near neutral (pH=about 8.5) at the time of use. But since a strong acid used for the dissolution hydrolyzes this substrate so as to raise the blank value gradually, a usable period of the formulation is about 5 hours after the preparation.
In order to improve the solubility of this substrate, various methods have been proposed and used practically as follows: (1) A method wherein a cationic surface active agent or anionic surface active agent is added to this substrate to improve the solubility of this substrate in water and an inhibitory action for the .gamma.-GTP activity by such an ionic surface active agent is relaxed by a nonionic surface active agent. (2) A method wherein a water-soluble group such as a --CO.sub.2 H group, --SO.sub.3 H group, or the like is provided at an ortho position relative to the --NO.sub.2 group of this substrate to improve the regarding solubility of this substrate in water. (3) A method wherein the solubility of this substrate in water is improved by applying clathrating force of cyclodextin (Japanese Patent Unexamined Publication No. 74099/82).
But these methods have various disadvantages. For example, according to the method (1) using a surface active agent, the inhibitory action of the ionic surface active agent for .gamma.-GTP is so strong that the recovery rate of .gamma.-GTP activity is about 70 to 80% even if a nonionic surface active agent is added. Further, by the addition of the surface active agent, the absorption of hemoglobin changes with the lapse of time and the absorption of 410 nm used for tracing the generating rate of p-nitroaniline is reduced with the lapse of time, so that a negative error is consequently given to the measured value of .gamma.-GTP activity. According to the method (2) for providing a water-soluble group, there are various problems in that measured values of .gamma.-GTP activity are higher than the true values, the substrate in a formulation solution is denatured in several days after the preparation to lower measured values of .gamma.-GTP activity, and sometimes a precipitate due to the denaturing is deposited. Further, according to the method (3) for applying the clathrating force of cyclodextrin, there is no problem of influence of hemoglobin and variation of measured values of .gamma.-GTP activity. But since the improvement of the solubility in water is insufficient, when a substrate solution containing cyclodextrin as a clathrating reagent is freeze dried, the concentration of the solution for preparations is that of the final use or twice of that at most.
In order to improve such a disadvantage of using cyclodextrin, there are proposed the use of modified cyclodextrins in place of cyclodextrin (Japanese Patent Unexamined Publication Nos. 160896/85 and 1399/86). According to Japanese Patent Unexamined Publication No. 160896/85, there are used cyclodextrins modified by introducing water-soluble groups such as NO.sub.2, PO.sub.3 H, SO.sub.3 H, COOH, etc. into the positions of hydroxyl groups. The solubility of the modified cyclodextrins at room temperature is improved, but clathrating force by the modified cyclodextrins at low temperatures is insufficient and crystals are deposited when allowed to stand at low temperatures for a long period of time. On the other hand, according to Japanese Patent Unexamined Publication No. 1399/86 wherein the hydroxyl groups of cyclodextrin is replaced by methyl groups, freeze dried products are poor in stability and reagent blank values rise when the modified cyclodextrins are used after being stored at room temperature for a long period of time.
On the other hand, as to a mechanism for forming clathrate compounds, there has been proposed a participation of various intermolecular force. It seems that a part or whole of various intermolecular forces such as dispersion force, interdipole force, hydrogen bond, hydrophobic bond, charge transfer force, etc., is involved in the formation of cyclodextrin clathrate compound. Therefore, if chemical structures of molecules are different, mechanisms for forming clathrate compounds are naturally different. Further, even if a clathrate compound is formed, there is a fear of suppressing the enzymatic reaction by making the whole of .gamma.-L-glutamyl-p-nitroanilide which is a guest molecule or active site for enzymatic reaction with .gamma.-GTP clathrated in the cyclodextrin cavity which is the host molecule. Therefore, even if .gamma.-L-glutamyl-p-nitroanilide is clathrated by cyclodextrin, it is impossible to expect whether other cyclodextrin derivatives can clathrate .gamma.-L-glutamyl-p-nitroanilide or not. Even if .gamma.-L-glutamyl-p-nitroanilide can be clathrated by the other cyclodextrin derivatives, it is impossible to expect whether the clathrated .gamma.-L-glutamyl-p-nitroanilide maintains a function as the substrate for .gamma.-GTP or not.